Dyskeratosis congenita (DKC) is a rare heritable disorder characterized by the triad of reticular skin pigmentation, nail dystrophy, and mucosal leukoplakia. Progressive bone marrow failure develops in more than 40% of the patients by the age of 10 y and in more than 80% of the patients by the age of 30 y, which is the main cause of early mortality (Knight et al. 1998). Several types of malignancies are also frequently complicated (Knight et al. 1998). The clinical features of DKC suggest that tissues in active renewal may be most vulnerable to this genetic abnormality.
DKC is a genetically heterogeneous disease (Dokal, 2000). The X-linked form accounts for most of the disease with more than 90% of the patients being males. Autosomal recessive and autosomal dominant DKC are only occasionally encountered. Mutations in DKC1 gene which encodes dyskerin were identified to be responsible for the X-linked DKC by positional cloning (Heiss et al. 1998). Since then a variety of DKC1 mutations have been characterized (Knight et al. 1999), however, previous reports of mutations did not accompany detailed description of their clinical features.
Here we report two Chinese pedigrees with the X-linked form of DKC. Five patients with typical mucocutaneous triad were found in the two families. A novel mutation of 1226C
T (P409L) and a de novo mutation of 1058CT (A353 V) in DKC1 were identified. This is the first report of DKC mutation analysis in Chinese patients.
RESULTS
For the three affected members (III:5, 9, and 13) of the first pedigree (Figure 1a), sequencing of the PCR products amplified from exon 12 of the DKC1 revealed a transition mutation of C to T in the coding region at nucleotide position 1226 (numbered from the A of the translation initiation codon ATG) (Figure 2a). Sequencing of the repeated PCR products from the opposite direction confirmed the mutation (Figure 2b). This missense mutation caused a replacement of the normal proline at amino acid residue 406 by leucine (P409L) in dyskerin. No mutation was found in the other exons or their flanking regions. Sequencing of the PCR products amplified from the patients' mothers (II:5, 7, and 9), maternal grandmothers (I:2), and one sister (III:12) showed heterozygous 1226CT mutation (Figure 2c). No such mutation was found in rest of the family members or in the 50 unrelated controls (Figure 2d). The BspMI digestion of the 227bp PCR product amplified from exon 12 yielded two fragments of 150 and 77 bp from the wild-type allele, and a 227 bp fragment from the mutant allele. As shown in Figure 1c, the BspMI digestion confirmed that the three male patients (III:5, 9, and 13) had one same mutant allele. Their mothers (II:5, 7, and 9), maternal grandmother (I:2), and one sister (III:12) carried one mutant allele and one wild-type allele. Their fathers (II:6 and 8) and the proband's sibling sister (III:14) had normal alleles.
Figure 1.
The two DKC pedigrees and mutation identification by endonuclease restriction enzyme digestion. Arrow, proband; star, DNA sample available; M, pBR322 DNA/MspI marker; N, normal control. (a) The first pedigree. (b) the second pedigree, (c) PCR products amplified from exon 12 of DKC1 were digested with BspMI and separated in polyacrylamide gel, and (d) PCR products from exon 11 of DKC1 were digested with MspA1 I and separated in polyacrylamide gel.
Full figure and legend (51K)Figure 2.
Direct sequencing of PCR products amplified from DKC1 gene (GenBank accession number NT-025965). Arrows indicate the positions of mutated bases. (a–d) Sequencing of PCR products from exon 12. (a) 1226CT mutation from the proband (III:13) of the first pedigree. (b) Sequencing from the opposite direction confirms the 1226CT mutation from the proband. (c) Heterozygous 1226CT mutation from family member II:9 of the first pedigree. (d) Sequencing of a normal control. (e–g): Sequencing of PCR products from exon 11. (e) 1058CT mutation from the proband (IV:2) of the second pedigree. (f) Heterozygous 1058CT mutation from family member III:7 of the second pedigree. (g) Sequencing of a normal control.
In the second pedigree (Figure 1b), the proband (IV:2) and his brother (IV:3) were identified to have a transition mutation of 1058CT (Figure 2e and Figure 1d) which resulted in an amino acid change of A353 V in dyskerin. Their mother (III:7) and sister (IV:1) carried a heterozygous 1058CT mutation (Figure 2f and Figure 1d), but their maternal grandparents (II:3 and 4) did not have this mutant allele (Figure 1d). Microsatellite markers proved that their mother (III:7) was the biological daughter of their grandparents (II:2 and 3), and the 1058CT mutation was therefore a de novo event beginning from proband's mother (III:7).
DISCUSSION
Symptoms of DKC usually begin during patients' childhood. Mucocutaneous features including pigmentation of skin, nail dystrophy, and leukoplakia appear between the ages of 5–10 y (Knight et al. 1998). Apart from the typical triad manifestations, a variety of other clinical findings have also been described (Dokal, 2000). Dysphagia, epiphora, and dysuria are common symptoms resulting from constriction of the mucosal surface of esophagus, lacrimal ducts, and urethra. Our five patients were diagnosed as DKC based on the clinical manifestations of reticulate pigmentation, nail dystrophy, and oral leukoplakia, although no other systemic involvements were found at the present time. DKC patients are usually complicated with hemotopoietic deficiency in the second or third decade of life, and malignancies including squamous cell carcinomas of skin or mucosa in the third or fourth decade (Dokal, 2000). Therefore, the patients should be closely followed.
DKC presents as an X-linked recessive, autosomal recessive or autosomal dominant inherited disease. Most of the DKC families are of X-linked recessive inheritance, with 90% of the DKC patients being males (Dokal, 2000). Mutations in the DKC1 gene are responsible for the X-linked DKC (Heiss et al. 1998). Autosomal dominant form of DKC is caused by mutations in the hTR gene (Vulliamy et al. 2001). In this study, all of the patients were males, whose parents and sisters presented with normal phenotype, suggesting the X-linked recessive inheritance in the two families. We therefore chose DKC1 for mutation search.
The DKC1 gene consists of 15 exons and spans a 15 kb region at Xq28. DKC1 encodes the nucleolar protein dyskerin and is ubiquitously expressed in tissues (Heiss et al. 1998; Hassock et al. 1999). Most mutations responsible for DKC are missense mutations in the coding regions of the gene (Heiss et al. 1998; Hassock et al. 1999; Knight et al. 1999). In addition, two putative splicing mutations in introns 1 and 2 (Knight et al. 1999; Knight et al. 2001), a single amino acid deletion (Heiss et al. 1998), a 2 kb deletion of exon 15 (Heiss et al. 1998), and a mutation in the upstream regulatory region within a putative Sp1 binding site (Knight et al. 2001) have also been described. No correlation, however, was found between the type of mutation in DKC1 and the phenotype of the disease. The substitution of 1226CT (P409L) found in patients in the first pedigree was not reported previously. In the first pedigree, the three patients' mothers and their maternal grandmother carried a heterozygous 1226CT mutation, however they were unaffected, indicating the typical X-linked recessive inheritance. The de novo 1058CT mutation as seen in our second pedigree has been reported several times. Therefore, the 1058CT mutation may be a hotspot for de novo mutation in DKC (Knight et al. 1999, 2001).
The pathological consequences of mutations in dyskerin remain unknown. Dyskerin is a putative pseudouridine synthase that localizes in a complex with box H/ACA small nucleolar RNAs and mediates posttranscriptional modification of ribosomal RNA through the conversion of uridine to pseudouridine (Heiss et al. 1998; Knight et al. 1999). Dyskerin is also associated with the RNA component of human telomerase that contains an H/ACA RNA motif (Mitchell et al. 1999a) and DKC was proposed to be caused by the defects in the maintenance of telomeres (Blasco et al. 1997; Mitchell et al. 1999b). Recent results from Dkc1 mutant mice suggest that ribosome malfunction is important in the initiation of DKC, whereas telomere shortening may modify and/or exacerbate DKC (Ruggero et al. 2003). Further studies of DKC patients are necessary to understand the role of mutant dyskerin in the pathogenesis of DKC and its hematological and malignant complications.
MATERIALS AND METHODS
Patients
In the first pedigree (Figure 1a), three patients were found among 26 members who came from the Hebei province in Northern China. The proband (III:13) was a 20-y-old Chinese male with skin lesions, nail dystrophy, and oral mucosa erosions since childhood. He developed epiphora, dysphagia, and dysuria during adolescence and the symptoms deteriorated with age. The mental and physical development was normal. Physical examination revealed generalized reticulation of hyperpigmentation, hypopigmentation, and fine telangiectatic erythema, prominently on face, trunk and limbs (Figure 3a). All of his nails were found dystrophic (Figure 3b and Figure 3c), and the finger tips became shiny and smooth. Erosions and dystrophy with patches of leukoplakia were found on his buccal mucosa, tongue (Figure 3d), and coronary sulcus of the penis (Figure 3e), however, the teeth were normal. Eye involvement included recurrent conjunctivitis and excessive lacrimation. Routine laboratory examinations including blood counts, urinalysis, serum electrolytes, hepatic and renal function tests, and immunoglobulin levels as well as chest X-ray and ECG were within normal limits. A punch biopsy taken from hyperpigmented lesions on the dorsal skin revealed atrophic epidermis with areas of hyperpigmentation in the basal layer; and melanophages, telangiectasia and mild infiltration of inflammatory cells in the superficial dermis. Blister forming was observed beneath the epidermis due to liquefaction degeneration of the basal cells (Figure 3f). The other two patients (III:5 and 9) were both 20 y old and had similar manifestations.
Figure 3.
Clinical data of the DKC proband in the first pedigree. (a) Hyperpigmentation, hypopigmentation, and fine telangiectatic erythema on the neck. (b, c) Dystrophy of nails. (d, e) Dystrophy with patches of leukoplakia on tongue and coronary sulcus of the penis. (f) Skin biopsy from a hyperpigmentation site shows atrophic epidermis with areas of hyperpigmentation in the basal layer, and melanophages, telangiectasia, and mild infiltration of inflammatory cells in the superficial dermis. Blister beneath the epidermis due to liquefaction degeneration of the basal cells. Scale bar=100 
m.
In the second pedigree (Figure 1b), two members were affected among 20 people. The proband (IV:2) was a 19-y-old male. He was normal until the age of 5 y when he developed erosion on oral mucosa followed by nail dystrophy. At the age of 10 y, he had skin dyspigmentation, palmoplantar hyperkeratosis, dysphagia, and epiphora. No other abnormalities were found. His younger brother (IV:3, 16 y old) had similar but less severe manifestations. Other living members in the two families were examined and no stigmata of DKC were found.
Mutation detection
The studies performed were approved by the Institutional Review Board of the Peking University First Hospital. With informed consent, we collected blood samples of attainable individuals in the two pedigrees as well as 50 unrelated normal individuals for controls. Genomic DNA was isolated from peripheral white blood cells. The coding exons of the DKC1 gene and the flanking regions (GenBank accession number NT025965) were amplified by polymerase chain reaction (PCR). The primers for the amplification of exon 11 were: forward primer 5'-tccatatgcacatcctgagc and reverse primer 5'-tccccctctgtgagaaacac. The primers for the amplification of exon 12 were: forward primer 5'-attctttgtag-tcaccatgcc and reverse primer 5'-agcaa gtgtgccgtctctacc. PCR products were purified by electrophoresis in agarose gel and sequenced using an automated sequencer (ABI Model 377, Applied Biosystems, Foster City, California). The mutations of 1226CT in exon 12 and 1058 CT in exon 11 eliminate the normal BspMI site and MspA1 I site, respectively. Mutations were further confirmed by digestion of PCR products with the two endonuclease restriction enzymes at 37°C for 4 h, electrophoresis in 8% polyacrylamide gel and staining with silver nitrate.
Paternity identification
To confirm the de novo mutation of 1058CT in the second pedigree (Figure 1b), the DNA samples from the proband's mother (III:7) and maternal grandparents (II:3 and 4) were sent to the Criminal Investigation Center of Beijing Public Security Bureau for paternity identification using the 16 highly polymorphic microsatellite markers and Identifiler Kit (Applied Biosystems, Foster City, California).
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Acknowledgments
We sincerely thank the DKC families for their cooperation in this study. This work was supported in part by Beijing Natural Science Fund (7012020).
